In the adult brain, glutamate is the principal excitatory transmitter that acts at central synapses. Despite the importance of glutamate, relatively little work has examined the release of and response to glutamate in early neuronal development, and the functional role of glutamate in early development. During this period, glutamate may play a key role in modulating synaptogenesis, neuronal architecture, cellular migration, gene expression, and neuronal survival. We will address the central hypothesis that glutamate plays a significant role as an intercellular messenger in early brain development, even before synapse formation. To determine if axonal growth cones release glutamate, thereby influencing potential postsynaptic target cells we will use outside-out and whole cell patch-clamp electrical recordings; membrane patches, taken from glutamate receptor-containing rat hippocampal neurons, or whole neurons, will be placed close to an axonal growth cone as an ultrasensitive approach to detect glutamate release in vitro. Parallel ultrastructural immunocytochemical experiments with colloidal gold will compare glutamate immunoreactivity in growing and differentiating axons, dendrites, and synapses. We will examine th ehypothesis that glutamate modulates rapid changes in dendritic morphology, possibly as a prelude to synapse formation, through a number of glutamate receptor mechznisms including ionotropic and metabotropic receptors, and voltage activated calcium channels. We will also examine membrane turnover and transmitter vesicle exocytosis in presumptive glutamatergic neurons with the dye FM1-43, studied with confocal microscopy to test the hypothesis thta membranes of growing axons increase exocytosis on contact with a postsynaptic neuron, and that exocytosis in developing neurons is modulated by glutamate stimulation. Using Northern blots and Western blots, we will test the hypothesis that glutamate released during development modulates the expression of specific glutamate receptor subtypes by culturing neurons with specific glutamate receptor blockers. This project addresses the fundamental importance of glutamate during neuronal development using converging morphological, immunocytochemical, physiological, and molecular approaches. Glutamate~s triple role as transmitter, toxin, and tropin makes it a crucial player both in normal development and in a number of human disorders. Glutamate dysfunction has been postulated to be a factor in learning disabilities, schizophrenia, epilepsy, secondary response to brain injury or high fever, ischemia, hypoxia, AIDS-related dementia, cerebral palsy, and Rasmussen~s encephalitis.